Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s^?1 is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

Viscosity based droplet size controlling in negative pressure driven droplets generator for large‐scale particle synthesis

The poor control and regulation of droplets limit the applications of negative pressure driven droplet generator (NPDDG). Here, we present a simple method to control droplet size in NPDDG via varying the oil viscosity. Depending on the infinite intersolubility of high viscous mineral oil and low viscous hexadecane, we obtain a series of oils with kinematic viscosities linearly varied from 4.2 to 194.6 mm²/s. By using these oils as continuous phases, monodisperse droplets are fabricated with controllable size in NPDDG. This viscosity‐based droplet regulation method is effective, reliable, and compatible with scale‐up processes. Compared with droplet generator driven by positive pressure, the presented method can fabricate hydrogel particles massively, without complicated multilayer chip structure and complex fluid controlling, which may extend the potential of NPDDG in droplets based high‐throughput assay or large‐scale materials synthesis.     

Optimal preparation methods for automated matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry profiling of low molecular weight proteins and peptides

Mass spectrometry (MS) profiling of the proteome and peptidome for disease‐associated patterns is a new concept in clinical diagnostics. The technique, however, is highly sensitive to external sources of variation leading to potentially unacceptable numbers of false positive and false negative results. Before MS profiling can be confidently implemented in a medical setting, standard experimental methods must be developed that minimize technical variance. Past studies of variance have focused largely on pre‐analytical variation (i.e., sample collection, handling, etc.). Here, we examined how factors at the analytical stage including the matrix and solid‐phase extraction influence MS profiling. Firstly, a standard peptide/protein sample was measured automatically by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS across five consecutive days using two different preparation methods, dried droplet and sample/matrix, of four types of matrix: α‐cyano‐4‐hydroxycinnamic acid (HCCA), sinapinic acid (SA), 2,5‐dihydroxybenzoic acid (DHB) and 2,5‐dihydroxyacetophenone (DHAP). The results indicated that the matrix preparation greatly influenced a number of key parameters of the spectra including repeatability (within‐day variability), reproducibility (inter‐day variability), resolution, signal strength, background intensity and detectability. Secondly, an investigation into the variance associated with C8 magnetic bead extraction of the standard sample prior to automated MS profiling demonstrated that the process did not adversely affect these same parameters. In fact, the spectra were generally more robust following extraction. Thirdly, the best performing matrix preparations were evaluated using C8 magnetic bead extracted human plasma. We conclude that the DHAP prepared according to the dried‐droplet method is the most appropriate matrix to use when performing automated MS profiling. Copyright © 2009 John Wiley & Sons, Ltd.     

A novel and rapid extraction protocol for sensitive and accurate determination of prochloraz in orange juice samples: Vortex‐assisted spraying‐based fine droplet formation liquid‐phase microextraction before gas chromatography–mass spectrometry

A novel, ecofriendly, and easy extraction and preconcentration method named as vortex‐assisted spraying‐based fine droplet formation liquid‐phase microextraction was proposed for the determination of prochloraz at trace levels in orange juice samples by gas chromatography–mass spectrometry (GC‐MS). In this novel system, extraction solvent is dispersed by the help of spraying apparatus instead of dispersive solvent. Various parameters of the method were carefully optimized to increase signal‐to‐noise ratio of the analyte. Under the optimum chromatographic and extraction conditions, limit of detection and limit of quantification were calculated as 3.2 and 10.8 μg/kg, respectively. Moreover, enhancement in quantification power for the GC‐MS system was determined as 372 folds based on LOQ comparison. Relative recovery results for orange juice samples were found to be between 95.0–107.7% by utilizing matrix matching calibration. Furthermore, the developed method may be used to efficiently and simply extract other organic compounds for their determinations in several matrices.     

N‐alkylpyridinium quaternization for assisting electrospray ionization of sterols in oil by quadrupole‐time of flight mass spectrometry

Rationale: The illegal cooking oil has become a serious social problem and raised widespread alarm recently. However, an efficient and sensitive technique for identifying the potential illegal cooking oil is still unavailable, especially when mixed with the ordinary ones; there is an urgent need to develop an efficient method for identifying the illegal cooking oil. Sterols in the cooking oil could be used as an indicator to identify the source and quality of oil by detecting the kinds of phytosterols and zoosterols. However, those sterols are difficult to be ionized by electrospray ionization, which resulted in the low sensitivity in electrospray ionization (ESI)‐mass spectrometric (MS) analysis. Methods: N‐alkylpyridinium isotope quaternization was extended to charge label sterols in different cooking oil and attached N‐cationic pyridinium tag onto the sterols in the presence of trifluoromethanesulfonic anhydride (Tf₂O); the kinds of sterols were identified and quantified by comparing d₀/d₅ pairs and product scan from ESI‐quadrupole‐time of flight (Q‐TOF) MS analysis. Results: The derivatized sterols were attached with permanent charge, resulting in the significant enhancement of ionization in ESI‐Q‐TOF MS analysis. The detection limits of analytes were improved to 0.02–0.05 ng/mL; different kinds of phytosterol, zoosterol and oxides were identified and quantified by comparing d₀/d₅ pairs from full scan and product scan. The method was applied in the detection of zoosterol for identifying the potential recycled cooking oil, even when the illegal oil has been blended into the ordinary one. More zoosterol was detected in the recycled oil compared with other cooking oil. Conclusions: The use of N‐alkylpyridinium isotope quaternization method provided an alternative method for identifying the potential illegal cooking oil. Copyright © 2013 John Wiley & Sons, Ltd.     

Light‐Directing Omnidirectional Circularly Polarized Reflection from Liquid‐Crystal Droplets

Constructing and tuning self‐organized three‐dimensional (3D) superstructures with tailored functionality is crucial in the nanofabrication of smart molecular devices. Herein we fabricate a self‐organized, phototunable 3D photonic superstructure from monodisperse droplets of one‐dimensional cholesteric liquid crystal (CLC) containing a photosensitive chiral molecular switch with high helical twisting power. The droplets are obtained by a glass capillary microfluidic technique by dispersing into PVA solution that facilitates planar anchoring of the liquid‐crystal molecules at the droplet surface, as confirmed by the observation of normal incidence selective circular polarized reflection in all directions from the core of individual droplet. Photoirradiation of the droplets furnishes dynamic reflection colors without thermal relaxation, whose wavelength can be tuned reversibly by variation of the irradiation time. The results provided clear evidence on the phototunable reflection in all directions.     

Synthesis of large,high‐solid‐content latexes by miniemulsion polymerization

Multiple and diverse applications have been recently found for miniemulsions and miniemulsion polymerization. In this work, miniemulsion polymerization is presented as a suitable technique for the preparation of high‐solid‐content latices with large particle sizes. Monomer miniemulsions were prepared with a high‐pressure homogenizer, and droplet sizes of 200–700 nm were obtained. Latexes with particle sizes larger than the sizes commonly accepted for miniemulsion polymerization were obtained. With fixed operational conditions of the homogenizer, the type of stabilizer was the key parameter determining the droplet size and the droplet size distribution. The particle size of the latices obtained by miniemulsion polymerization indicated that the particles were mainly formed by droplet nucleation. Latexes obtained by this process have multiple applications, including use as seeds in the polymerization of high‐solid‐content latices. This article shows that potential new applications for miniemulsion polymerization are far from being exhausted. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4222–4227, 2004     

Quantification of triacylglycerol regioisomers by ultra‐high‐performance liquid chromatography and ammonia negative ion atmospheric pressure chemical ionization tandem mass spectrometry

The regioisomer composition of triacylglycerols (TAGs) in various vegetable oils was determined with a new liquid chromatography/tandem mass spectrometry (LC/MS/MS method). A direct inlet ammonia negative ion chemical ionization (NICI) MS/MS method was improved by adapting it to LC negative ion (NI) atmospheric pressure chemical ionization (APCI) MS/MS system using ammonia as nebulizer gas. The method is based on the preferential formation of [M–H–RCOOH–100]^? ions during collision‐induced dissociation by loss of sn‐1/3 fatty acids from [M–H]^? ions. Calibration curves were created from nine reference TAGs: Ala/L/L, Gla/L/L, L/L/O, L/O/O, P/O/O, P/P/O, Po/Po/V, Po/Po/O, and C/O/O. The calibration curves were used to quantify the regioisomer compositions of selected TAGs in rapeseed oil, sunflower seed oil, palm oil, black currant seed oil, and sea buckthorn pulp oil. The method discriminates the different regioisomers and the results obtained by this method were in good agreement with previous results. This proves that this new method can be used for the determination of regiospecific distribution of fatty acids in TAGs. Copyright © 2009 John Wiley & Sons, Ltd.     

Chemical and Topographical Single‐Cell Imaging by Near‐Field Desorption Mass Spectrometry

Simultaneously acquiring chemical and topographical information within a single cell at nanoscale resolutions is vital to cellular biology, yet it remains a great challenge due to limited lateral resolutions and detection sensitivities. Herein, the development of near‐field desorption mass spectrometry for correlated chemical and topographical imaging is reported, thereby bridging the gap between laser‐based mass spectrometry (MS) methods and multimodal single‐cell imaging. Using this integrated platform, an imaging resolution of 250 nm and 3D topographically reconstructed chemical single‐cell imaging were achieved. This technique offers more in‐depth cellular information than micrometer‐range laser‐based MS imaging methods. Considering the simplicity and compact size of the near‐field device, this technique can be introduced to MALDI‐MS, expanding the multimodal abilities of MS at nanoscale resolutions.     

Flexible particle flow‐focusing in microchannel driven by droplet‐directed induced‐charge electroosmosis

We report herein a novel microfluidic particle concentrator that utilizes constriction microchannels to enhance the flow‐focusing performance of induced‐charge electroosmosis (ICEO), where viscous hemi‐spherical oil droplets are embedded within the mainchannel to form deformable converging‐diverging constriction structures. The constriction region between symmetric oil droplets partially coated on the electrode strips can improve the focusing performance by inducing a granular wake flow area at the diverging channel, which makes almost all of the scattered sample particles trapped within a narrow stream on the floating electrode. Another asymmetric droplet pair arranged near the outlets can further direct the trajectory of focused particle stream to one specified outlet port depending on the symmetry breaking in the shape of opposing phase interfaces. By fully exploiting rectification properties of induced‐charge electrokinetic phenomena at immiscible water/oil interfaces of tunable geometry, the expected function of continuous and switchable flow‐focusing is demonstrated by preconcentrating both inorganic silica particles and biological yeast cells. Physical mechanisms responsible for particle focusing and locus deflection in the droplet‐assisted concentrentor are analyzed in detail, and simulation results are in good accordance with experimental observations. Our work provides new routes to construct flexible electrokinetic framework for preprocessing on‐chip biological samples before performing subsequent analysis.     

Open Probe fast GC–MS — combining ambient sampling ultra‐fast separation and in‐vacuum ionization for real‐time analysis

An Open Probe inlet was combined with a low thermal mass ultra‐fast gas chromatograph (GC), in‐vacuum electron ionization ion source and a mass spectrometer (MS) of GC–MS for obtaining real‐time analysis with separation. The Open Probe enables ambient sampling via sample vaporization in an oven that is open to room air, and the ultra‐fast GC provides ~30‐s separation, while if no separation is required, it can act as a transfer line with 2 to 3‐s sample transfer time. Sample analysis is as simple as touching the sample, pushing the sample holder into the Open Probe oven and obtaining the results in 30 s. The Open Probe fast GC was mounted on a standard Agilent 7890 GC that was coupled with an Agilent 5977A MS. Open Probe fast GC–MS provides real‐time analysis combined with GC separation and library identification, and it uses the low‐cost MS of GC–MS. The operation of Open Probe fast GC–MS is demonstrated in the 30‐s separation and 50‐s full analysis cycle time of tetrahydrocannabinol and cannabinol in Cannabis flower, sub 1‐min analysis of trace trinitrotoluene transferred from a finger onto a glass surface, vitamin E in canola oil, sterols in olive oil, polybrominated flame retardants in plastics, alprazolam in Xanax drug pill and free fatty acids and cholesterol in human blood. The extrapolated limit of detection for pyrene is <1 fg, but the concentration is too high and the software noise calculation is untrustworthy. The broad range of compounds amenable for analysis is demonstrated in the analysis of reserpine. The possible use with alternate standard GC–MS and Open Probe fast GC–MS is demonstrated in the analysis of heroin in its street drug powder. The use of Open Probe with the fast GC acting as a transfer line is demonstrated in <10‐s analysis without separation of ibuprofen and estradiol. Copyright © 2017 John Wiley & Sons, Ltd.     

Organization of nucleobase‐functionalized β‐peptides investigated by soft electrospray ionization mass spectrometry

The development and validation of analytical methods is a key to succeed in investigating noncovalent interactions between biomolecules or between small molecules and biomolecules. Electrospray ionization mass spectrometry (ESI‐MS) was applied with a Fourier transform ion cyclotron resonance mass spectrometer (FTICR‐MS) as well as a quadrupole/time‐of‐flight tandem mass spectrometer (QqToF‐MS) for a systematic investigation of noncovalent complexes based on nucleobase pairing in an artificial and noncharged backbone topology. Synthetical β‐peptide helices covalently modified with nucleobases were organized by recognition of a sequence of four nucleobases. Specific duplexes of β‐peptide helices were obtained on the basis of hydrogen bonding base pair complementarity. Oligomer interactions were detected with defined stoichiometry and sensitivity for the respective duplex stability. FTICR‐MS and QqToF‐MS were used equally well to indicate double strand stabilities in agreement with the dissociation data determined by UV spectroscopy. Furthermore, the dissociation energies of gas phase ions of the noncovalent complexes were analyzed with collision induced dissociation (CID)‐MS/MS and infrared multiphoton dissociation (IRMPD)‐MS/MS. The CID conditions turned out to be too harsh for a differentiation of the duplex stabilities, whereas IRMPD might be developed as a technique to detect even small interaction energy differences. Copyright © 2009 John Wiley & Sons, Ltd.     

The application of gas chromatography/atmospheric pressure chemical ionisation time‐of‐flight mass spectrometry to impurity identification in Pharmaceutical Development

Accurate mass measurement (used to determine elemental formulae) is an essential tool for impurity identification in pharmaceutical development for process understanding. Accurate mass liquid chromatography/mass spectrometry (LC/MS) is used widely for these types of analyses; however, there are still many occasions when gas chromatography (GC)/MS is the appropriate technique. Therefore, the provision of robust technology to provide accurate mass GC/MS (and GC/MS/MS) for this type of activity is essential. In this report we describe the optimisation and application of a newly available atmospheric pressure chemical ionisation (APCI) interface to couple GC to time‐of‐flight (TOF) MS. To fully test the potential of the new interface the APCI source conditions were optimised, using a number of standard compounds, with a variety of structures, as used in synthesis at AstraZeneca. These compounds were subsequently analysed by GC/APCI‐TOF MS. This study was carried out to evaluate the range of compounds that are amenable to analysis using this technique. The range of compounds that can be detected and characterised using the technique was found to be extremely broad and include apolar hydrocarbons such as toluene. Both protonated molecules ([M + H]⁺) and radical cations (M^+.) were observed in the mass spectra produced by APCI, along with additional ion signals such as [M + H + O]⁺. The technique has been successfully applied to the identification of impurities in reaction mixtures from organic synthesis in process development. A typical mass accuracy of 1–2 mm/zunits (m/z 80–500) was achieved allowing the reaction impurities to be identified based on their elemental formulae. These results clearly demonstrate the potential of the technique as a tool for problem solving and process understanding in pharmaceutical development. The reaction mixtures were also analysed by GC/electron ionisation (EI)‐MS and GC/chemical ionisation (CI)‐MS to understand the capability of GC/APCI‐MS relative to these two firmly established techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of two‐dimensional gas chromatography (GC×GC) coupled with Orbitrap‐technology‐based mass spectrometry: Interest in the identification of biofuel composition

Comprehensive gas chromatography (GC) has emerged in recent years as the technique of choice for the analysis of volatile and semivolatile compounds in complex matrices. Coupling it with high‐resolution mass spectrometry (MS) makes a powerful tool for identification and quantification of organic compounds. The results obtained in this study showed a significant improvement by using GC×GC‐EI‐MS in comparison with GC‐EI‐MS; the separation of chromatogram peaks was highly improved, which facilitated detection and identification. However, the limitation of Orbitrap mass analyzer compared with time‐of‐flight analyzer is the data acquisition rate; the frequency average was about 25 Hz at a mass resolving power of 15.000, which is barely sufficient for the proper reconstruction of the narrowest chromatographic peaks. On the other hand, the different spectra obtained in this study showed an average mass accuracy of about 1 ppm. Within this average mass accuracy, some reasonable elemental compositions can be proposed and combined with characteristic fragment ions, and the molecules can be identified with precision. At a mass resolving power of 7.500, the scan rate reaches 43 Hz and the GC×GC‐MS peaks can be represented by more than 10 data points, which should be sufficient for quantification. The GC×GC‐MS was also applied to analyze a cellulose bio‐oil sample. Following this, a highly resolved chromatogram was obtained, allowing EI mass spectra containing molecular and fragment ions of many distinct molecules present in the sample to be identified.     

MALDI‐FT‐ICR‐MS for archaeological lipid residue analysis

Soft‐ionization methods are currently at the forefront of developing novel methods for analysing degraded archaeological organic residues. Here, we present little‐used soft ionization method of matrix assisted laser desorption/ionization‐Fourier transform‐ion cyclotron resonance‐mass spectrometry (MALDI‐FT‐ICR‐MS) for the identification of archaeological lipid residues. It is a high‐resolution and sensitive method with low limits of detection capable of identifying lipid compounds in small concentrations, thus providing a highly potential new technique for the analysis of degraded lipid components. A thorough methodology development for analysing cooked and degraded food remains from ceramic vessels was carried out, and the most efficient sample preparation protocol is described. The identified components, also controlled by independent parallel analysis by gas chromatography‐mass spectrometry (GC‐MS) and gas chromatography‐combustion‐isotope ratio mass spectrometry (GC‐C‐IRMS), demonstrate its capability of identifying very different food residues including dairy, adipose fats as well as lipids of aquatic origin. The results obtained from experimentally cooked and original archaeological samples prove the suitability of MALDI‐FT‐ICR‐MS for analysing archaeological organic residues. Sample preparation protocol and identification of compounds provide future reference for analysing various aged and degraded lipid residues in different organic and mineral matrices.     

Development of a sheathless CE‐ESI‐MS interface

A sheath‐flow interface is the most common ionization technique in CE‐ESI‐MS. However, this interface dilutes the analytes with the sheath liquid and decreases the sensitivity. In this study, we developed a sheathless CE‐MS interface to improve sensitivity. The interface was fabricated by making a small crack approximately 2 cm from the end of a capillary column fixed on a plastic plate, and then covering the crack with a dialysis membrane to prevent metabolite loss during separation. A voltage for CE separation was applied between the capillary inlet and the buffer reservoir. Under optimum conditions, 52 cationic metabolite standards were separated and selectively detected using MS. With a pressure injection of 5 kPa for 15 s (ca. 1.4 nL), the detection limits for the tested compounds were between 0.06 and 1.7 μmol/L (S/N = 3). The method was applied to analysis of cationic metabolites extracted from a small number (12 000) of cancer cells, and the number of peaks detected was about 2.5 times higher than when using conventional sheath‐flow CE‐MS. Because the interface is easy to construct, it is cost‐effective and can be adapted to any commercially available capillaries. This method is a powerful new tool for highly sensitive CE‐MS‐based metabolomic analysis.     

A Micromechanical Cantilever‐Based Liposome Biosensor for Characterization of Protein‐Membrane Interaction

We have newly evaluated the interaction of lipid membrane with two different proteins of lysozyme and carbonic anhydrase from bovine (CAB) using a micro cantilever‐based liposome biosensor with a new droplet‐sealing structure. Herein 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) liposomes are used as model lipid membrane and are immobilized on the surface of cantilever. The interaction of DPPC liposome with the target protein causes deflection of the micro‐cantilever, which can stably be detected by measuring the resistance change of the strain gauge. The resistance change dependent on time is used to evaluate the characteristic of liposome‐protein interaction. The resistance of the cantilever‐based biosensor increases monotonously with time in both of the two protein solutions. Especially, chronological resistance change depends markedly on both the concentration and species of target proteins. Finally, these results lead us to conclude that the cantilever‐based liposome biosensor with the droplet‐sealing structure facilitates the characterization of protein‐membrane interaction. It also means that this biosensor is a promising candidate device for label‐free detection of concentration and species of different target proteins.     

Confirmation of drug delivery after liver chemoembolization: direct tissue doxorubicin measurement by UHPLC‐MS‐MS

Because liver cancer is rarely suitable for surgery, transcatheter arterial chemoembolization (TACE) is used for palliative therapy. In this procedure, an emulsion of doxorubicin in iodized oil is injected directly into liver tumors through a catheter positioned within the artery supplying blood flow to the tumor. At present, there is limited understanding of factors affecting the delivery and dispersion of doxorubicin within treated tumors during TACE. This study addresses the development and application of an ultrahigh‐pressure liquid chromatography–tandem mass spectrometry (UHPLC‐MS‐MS) method for rapid confirmation of drug delivery after TACE in a rabbit VX2 liver cancer model. Doxorubicin levels in liver tumors were measured using UHPLC‐MS‐MS and compared with computed tomography measured levels of iodized oil, a metric used clinically to indicate drug delivery. We found that tissue drug levels determined using UHPLC‐MS‐MS did not correlate with the regional iodized oil concentration (vehicle) within tumors following TACE, suggesting that chemotherapeutic drugs like doxorubicin spread throughout tumors, and that lack of iodized oil staining in portions of a tumor does not necessarily indicate inadequate therapy during TACE. Copyright © 2012 John Wiley & Sons, Ltd.